Elsevier

Acta Tropica

Volume 149, September 2015, Pages 86-93
Acta Tropica

Immunological cross-reactivity and neutralization of the principal toxins of Naja sumatrana and related cobra venoms by a Thai polyvalent antivenom (Neuro Polyvalent Snake Antivenom)

https://doi.org/10.1016/j.actatropica.2015.05.020Get rights and content

Highlights

  • Immunological reactivity of NPAV against N. sumatrana venom toxins was examined.

  • The NPAV antivenom was moderately effective against PLA2 and long neurotoxins.

  • It was only weakly effective in neutralizing the short neurotoxin and cardiotoxin.

  • A‘proper-mix’ of cobra venoms as immunogen may help to improve antivenom efficacy.

Abstract

The low potency of cobra antivenom has been an area of concern in immunotherapy for cobra envenomation. This study sought to investigate factors limiting the neutralizing potency of cobra antivenom, using a murine model. We examined the immunological reactivity and neutralizing potency of a Thai polyvalent antivenom against the principal toxins of Naja sumatrana (Equatorial spitting cobra) venom and two related Asiatic cobra venom α-neurotoxins. The antivenom possesses moderate neutralizing potency against phospholipases A2 (P, potency of 0.98 mg/mL) and moderately weak neutralizing potency against long-chain α-neurotoxins (0.26–0.42 mg/mL) but was only weakly effective in neutralizing the short-chain α-neurotoxins and cardiotoxins (0.05–0.08 mg/mL). The poor neutralizing potency of the antivenom on the low molecular mass short-chain neurotoxins and cardiotoxins is presumably the main limiting factor of the efficacy of the cobra antivenom. Our results also showed that phospholipase A2, which exhibited the highest ELISA reactivity and avidity, was most effectively neutralized, whereas N. sumatrana short-chain neurotoxin, which exhibited the lowest ELISA reactivity and avidity, was least effectively neutralized by the antivenom. These observations suggest that low immunoreactivity (low ELISA reactivity and avidity) is one of the reasons for poor neutralization of the cobra venom low molecular mass toxins. Nevertheless, the overall results show that there is a lack of congruence between the immunological reactivity of the toxins toward antivenom and the effectiveness of toxin neutralization by the antivenom, indicating that there are other factors that also contribute to the weak neutralization capacity of the antivenom. Several suggestions have been put forward to overcome the low efficacy of the cobra antivenom. The use of a ‘proper-mix’ formulation of cobra venoms as immunogen, whereby the immunogen mixture used for hyperimmunization contains a mix of various types of α-neurotoxins and cardiotoxins in sufficient amount, may also help to improve the efficacy and broaden the neutralization spectrum of the antivenom.

Introduction

In Asia, cobra (Naja sp.) bites constitute the major cause of high mortality and morbidity associated with snake envenomation (Warrell, 2010, World Health Organization, 2010). Progressive descending paralysis and local necrosis are the prominent neurological manifestation of cobra envenomation. The early signs of muscular paralysis usually begin with ptosis, diplopia, ophthalmoplegia, dysphasia and dysphagia, followed by limb weakness and areflexia, eventually progress to respiratory failure as a consequence of intercostal muscles and diaphragm paralysis (Ahmed et al., 2008, Alirol et al., 2010). Respiratory failure occurs in 10–50% of cases (Punde, 2005, Wongtongkam et al., 2005) and is the leading cause of death in cobra envenomation (Wongtongkam et al., 2005).

Antivenom therapy has hitherto been the only definitive treatment for snake envenomation. Unfortunately, numerous studies have reported that most of the existing commercially available Asiatic cobra antivenoms are generally low in potency (Balde et al., 2013, Kularatne et al., 2009). The low neutralizing potency of Asiatic cobra antivenom (typically <2 mg venom/mL antivenom) implies that a very large amount (>20 vials) of the antivenom may be required to treat severe cobra envenomation, as the amount of venom injected by a cobra in a bite may range from 100 to 600 mg (Mirtschin et al., 2006). Administration of a very large amount of antivenom is, however, undesirable as it not only results in a very high treatment cost but also greatly increases the risk of hypersensitivity.

The present study was undertaken as part of an effort to investigate factors limiting the neutralizing and cross-neutralizing potency of Asiatic cobra antivenom. Cross-neutralization of Naja venoms by heterologous antivenom is a well-known phenomemon (Cham et al., 2013, Kornhauser et al., 2013, Leong et al., 2012a). Neuro Polyvalent Snake Antivenom (NPAV) (produced against the venoms of the Thai monocled cobra, Naja kaouthia, and three other Thai elapid snakes) was chosen for this study as it is a commonly used polyvalent antivenom in Thailand and Malaysia, and extensive cross-neutralization data of this antivenom against venoms from various cobras are available for comparison purpose (Leong et al., 2012a). This study investigated the immunoreactivity and neutralization profiles of NPAV against various principal toxins of Naja sumatrana (Equatorial spitting cobra), Naja kaouthia (Monocled cobra) and Naja sputatrix (Javen spitting cobra) venoms. Findings from the current study may provide clues to formulation of a more potent and efficacious cobra antivenom. This is particularly relevant for the optimization of paraspecific antivenom use on species like N. sumatrana (a Category 1 medically important snake in the region), the bite from which unfortunately has no species-specific antivenom available as treatment. Besides, the findings will also reveal if there is a good correlation between immunoreactivity and neutralization potency of cobra antivenom, as earlier studies have demonstrated that immunoreactivity of an antivenom does not necessarily reflect the in vivo neutralizing potency of the antivenom (Casewell et al., 2010).

Section snippets

Animals

Albino mice (ICR strain, 20–25 g) were supplied by the Laboratory Animal Centre, Faculty of Medicine, University of Malaya. The animals were handled according to the guidelines given by CIOMS on animal experimentation (Howard-Jones, 1985). The experimental protocols for all animal studies were approved by the Animal Care and Use Committee (ACUC) of the Faculty of Medicine, University of Malaya (Ethics Reference no. 2013-06-07/MOL/R/FSY). The institutional ethics committee was informed about the

Isolation of the principal toxins of N. sumatrana venom and neurotoxins from N. kaouthia and N. sputatrix venoms

The chromatographic profiles of N. sumatrana venom and its principal toxins (not shown) are similar to those reported by Yap et al. (2014). Six principal toxins were isolated from N. sumatrana venom, including one short-chain neurotoxin (sumatrana SNTX), one long-chain neurotoxins (sumatrana LNTX-II), two phospholipases A2 (sumatrana PLA2-I and sumatrana PLA2-II), and two cardiotoxins (sumatrana CTX-I and sumatrana CTX-II) (Yap et al., 2014).

Fig. 1 shows the isolation of N. kaouthia long-chain

Isolation of the cobra venom toxins

Yap et al. (2014) reported that eight of the N. sumatrana venom toxins constituted the major toxins of the venom, accounting for >90% of the total venom proteins. We selected the six most abundant toxins for this study. These include the two types of cobra venom α-neurotoxins: long and short-chain neurotoxins, and two each of the venom’s phospholipases A2 and cardiotoxins. The short-chain neurotoxin belongs to the type I α-neurotoxin subfamily, comprising 60–62 amino acid residues, while the

Conclusions

Our results suggest that there are major challenges to improving the potency of cobra antivenom, as the limitations to neutralization potency include both immunological characteristics of cobra venom toxins as well as non-immunological issues. The main cobra venom 3-finger toxins (α-neurotoxins and cardiotoxins) are generally poorly neutralized by the antivenom and poorly immunogenic too. The possible means to improve the effectiveness of neutralization include improving the avidity of

Acknowledgements

This work was supported by UM High Impact Research Grant UM.C/625/1/HIR/MOE/E00040-20001 from the Ministry of Education Malaysia, UMRG RG282-14AFR from University of Malaya, Malaysia and Science Fund, MOSTI, Government of Malaysia.

References (40)

  • F.W. Studier

    Analysis of bacteriophage T7 early RNAs and proteins on slab gels

    J. Mol. Biol.

    (1973)
  • N.H. Tan

    Isolation and characterization of two toxins from the venom of the Malayan cobra (Naja naja sputatrix)

    Toxicon

    (1983)
  • N.H. Tan

    Improvement of Malayan cobra (Naja naja sputatrix) antivenin

    Toxicon

    (1983)
  • M.K.K. Yap et al.

    Proteomic characterization of venom of the medically important Southeast Asian Naja sumatrana (Equatorial spitting cobra)

    Acta Trop.

    (2014)
  • S. Ahmed et al.

    Emergency treatment of a snake bite: pearls from literature

    J. Emerg. Trauma Shock

    (2008)
  • E. Alirol et al.

    Snake bite in South Asia: a review

    PLoS Negl. Trop. Dis.

    (2010)
  • M.C. Balde et al.

    Use of antivenoms for the treatment of envenomation by Elapidae snakes in Guinea, Sub-Saharan Africa

    J. Venomous Anim. Toxins Incl. Trop. Dis.

    (2013)
  • V. Brade et al.

    Immunization against cobra venom

    Experientia

    (1971)
  • N.R. Casewell et al.

    Pre-clinical assays predict pan-African Echis viper efficacy for a species-specific antivenom

    PLoS Negl. Trop. Dis.

    (2010)
  • G. Cham et al.

    Cross-reactivity against Naja sumatrana (Block spitting cobra) envenoming from the Haffkine antivenom in a mouse model

    ISRN Toxicology

    (2013)
  • Cited by (42)

    • Secretory phospholipase A<inf>2</inf> in snake venom and revelation from cobra venomics

      2023, Phospholipases in Physiology and Pathology: Volumes 1-7
    • Proteomic insights into short neurotoxin-driven, highly neurotoxic venom of Philippine cobra (Naja philippinensis) and toxicity correlation of cobra envenomation in Asia

      2019, Journal of Proteomics
      Citation Excerpt :

      As with the acidic PLA2 from N. kaouthia and N. naja venoms, the major PLA2 of N. philippinensis venom was found to be non-lethal up to 5 μg/g in mice, a dose that was ~25-time higher than the venom LD50 (0.18 μg/g) in this study. In contrast, the basic and neutral venom PLA2 of spitting cobras N. sputatrix [16] and N. sumatrana [59], respectively, are known to be lethal (LD50 = 0.5–2.0 μg/g). The biological activity of the acidic PLA2 could be associated with local effect (venom ophthalmia and tissue necrosis), and potentiation of cytotoxic activity for defensive as well as digestive purposes [5,60].

    View all citing articles on Scopus
    View full text